JP2013193985A - Hair tonic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hair tonic that promotes hair growth and hair-fostering and is effective for various kinds of alopecia.SOLUTION: There are provided a hair tonic that includes as an effective ingredient a plant selected from Anthriscus sylvestris Hoffm. and Crinum asiaticum L.var. sinicum Bak. or an extract thereof, and an agent for suppressing DACH1 expression or suppressing functions of DACH1.

Description

  The present invention relates to a hair restoring agent that exhibits a hair nourishing and hair restoring effect.

  In many cases of alopecia such as male pattern alopecia and alopecia areata, the details of the onset mechanism are still unclear. Conventionally, drugs such as blood circulation promoters, immunosuppressants, metabolic promoters, vitamins, and androgens have been used for the treatment of these alopecias.

  However, these drugs often have different effects depending on the symptoms and constitution, and the effects are not yet satisfactory. In addition, when used in a large amount, there are cases where an uncomfortable irritation sensation is given to an adaptation site, or dermatitis occurs due to continuous use.

  On the other hand, it is reported that Shak, a plant belonging to the genus Ceramaceae, is used as a folk remedy for dyspepsia, nourishing tonic, pollakiuria, and as an overnutrition absorption inhibitor. (Patent Document 1). Further, Taiwan Hamaomoto, which is a plant belonging to the genus Hamanomoto, has been used as a folk medicine for a long time, and has been reported to have a moisturizing effect, a whitening effect, and a neutral fat accumulation suppressing effect (Patent Document 2).

  However, it has not been known so far that sharks and Taiwan Hamaomoto have hair growth.

JP-A-8-217689 JP 2000-215566 A

  The present invention relates to providing hair growth agents that promote hair growth and hair growth and are effective in various alopecia.

  The present inventors have found that protein expression of DACH1 (DACHSHUND, DROSOPHILA, HOMOLOG OF, 1) is observed in the upper part of the hair bulb in hair tissue, and that growth of hair matrix cells is suppressed at the DACH1 expression site. It was. That is, it was found that DACH1 negatively regulates the growth of hair matrix cells in hair tissue, and that a substance that suppresses the expression and function of DACH1 has a hair growth promoting action. Further investigations have led to the finding that shaku and taiwan hamaomoto have the action of reducing the function of DACH1, and that these can serve as hair restorers.

That is, the present invention relates to the following 1) to 2).
1) A hair-growth agent comprising as an active ingredient a plant selected from shaku and taiwan hamaomoto or an extract thereof.
2) A DACH1 expression or function inhibitor comprising, as an active ingredient, a plant selected from shark and Taiwan Hamamoto or an extract thereof.

  By applying the hair restorer of the present invention to the scalp and the like, excellent hair growth / hair growth / hair growth action is exhibited at the application site.

Immunohistochemical staining showing the site of DACH1 expression in hair tissue. The graph which shows the growth inhibitory effect of the cultured cell by DACH1 overexpression. DACH1 (-): When not overexpressing DACH1. DACH1 (+): When DACH1 is overexpressed. The graph which shows AP-1 transcriptional activity suppression effect by DACH1 overexpression. The graph which shows the DACH1 function inhibitory effect of a shark and a Taiwan Hamamoto extract. DACH1 (-): When not overexpressing DACH1. DACH1 (+): When DACH1 is overexpressed. The graph which shows the human hair elongation promotion effect of a shark extract.

In the present invention, the term “saku” refers to Antriscus sylvestris (L.) Hoffm. "Taiwan Hamaomoto" is a genus Clanum asiaticum L. var. sinicum Bak. Means.

  The plant of the present invention can be used for whole plants, leaves, stems, buds, flowers, buds, roots, rhizomes, seeds, fruits, or the like, or a combination thereof. As for Hamamoto, the leaves are preferably used as they are or after being pulverized. Such a plant can be used as it is or as a squeezed juice obtained by squeezing it, a dried product obtained by drying the plant itself or a pulverized product thereof, or an extract extracted therefrom. Is preferred.

As the extract, various solvent extracts obtained by known extraction methods such as extraction of plants at room temperature or under heating, or extraction using an extractor such as a Soxhlet extractor, dilutions thereof, and concentration thereof A liquid or its dry powder is mentioned.
Known extraction methods include, for example, immersion, decoction, leaching, reflux extraction, supercritical extraction, ultrasonic extraction, and microwave extraction.

  As the extraction solvent used for obtaining the extract, either a polar solvent or a nonpolar solvent can be used. For example, water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; tetrahydrofuran and diethyl ether Linear and cyclic ethers such as polyethylene; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether; aroma such as benzene and toluene Group hydrocarbons; pyridines and the like, and these can be used alone or as a mixture. Of these, water, alcohol-based (alcohols and / or polyhydric alcohols) solvents, and water-alcohol mixed solvents are particularly preferable, and water-alcohol mixed solvents are preferable. Furthermore, as alcohols, methanol, ethanol, propanol (preferably isopropanol) and butanol (preferably 1-butanol) are more preferable, and ethanol is more preferable. The polyhydric alcohol is more preferably butylene glycol (preferably 1,3-butylene glycol).

  The plant extract of the present invention can be obtained, for example, by using 1 to 50 parts by mass of an extraction solvent with respect to 1 part by mass of the plant body and extracting at 4 to 100 ° C. for 0.5 hours to 30 days. . More specifically, when water is used as the extraction solvent, 5 to 30 parts by mass and 40 to 100 ° C. for 1 hour to 1 day are preferable with respect to 1 part by mass of the plant body. Moreover, when using a water-ethanol mixed solvent as an extraction solvent, 5-30 mass parts with respect to 1 mass part of plant bodies, 1 hour-20 days under room temperature-recirculation | reflux are preferable. Further, these operations may be repeated.

  The above extract can be used as it is, but it can also be used by diluting, concentrating or lyophilizing the extract and preparing it in a powder or paste form.

  Moreover, you may use the plant of this invention, or its extract in mixture of 2 or more types. Moreover, a commercial item may be used for an extract other than the said extraction processed material.

  The extract is preferably used after removing inactive impurities by a known technique such as liquid-liquid distribution, solid-liquid distribution, filtration membrane, activated carbon, adsorption resin, ion exchange resin, and the like. As the solvent used at this time, those exemplified for the extraction solvent may be used. Moreover, you may use these, after giving processes, such as a deodorizing and a decoloring, by a well-known method as needed. Moreover, when the extract is further purified, the known techniques and methods may be used.

As will be described later in Examples, the plant extract of the present invention has an action of reducing the function of DACH1 and has an effect of promoting hair elongation in a human hair follicle organ culture system. As shown in a reference example described later, expression of DACH1 is observed in the upper part of the hair bulb of hair tissue, and DACH1 is negative at a positive site of Ki67, which is a cell proliferation marker (FIG. 1). In addition, when DACH1 is overexpressed, the growth of human cultured cells (293A (human kidney) cells) is suppressed (FIG. 2). Therefore, it is considered that DACH1 negatively regulates the growth of hair matrix cells in hair tissue rich in its expression.
Therefore, hair growth can be promoted by suppressing (including inhibiting) the expression of DACH1 gene and the expression or function of DACH1 (Examples 1 and 2).
Therefore, the plant of the present invention or an extract thereof can be a hair restorer, or an expression or function inhibitor of DACH1 (referred to as “hair restorer etc.”), and can be used for producing a hair restorer or the like. . That is, it can be applied to humans and used for hair growth or for suppressing the expression or function of DACH1.

  The hair restorer itself may be cosmetic, quasi-drug, or pharmaceutical for hair growth or DACH1 expression or function suppression, or may be incorporated into the cosmetic, quasi-drug, pharmaceutical, etc. It may be a material or a preparation used.

Here, “hair growth” means, for example, at least one of hair growth, promotion of hair growth and prevention of hair loss, and is a concept including hair growth, promotion of hair growth (elongation), or hair growth.
Moreover, suppression of the expression of DACH1 includes suppression of the expression level of the DACH1 gene or DACH1, and suppression of the function of DACH1 includes, for example, c-Jun (one of the components of the AP-1 complex). Functions of binding and inhibiting the transcriptional activity of AP-1 and suppressing downstream gene expression (Wu K, et al. Mol Cell Biol. 26: 7116-7129. (2006)., Wu K, et al. Mol Biol Cell. 18: 755-767. (2007), Nan F, et al. Cancer Biol Ther. 8: 1534-1539. (2009).).

When the hair restorer of the present invention is used as cosmetics, quasi drugs or pharmaceuticals, it is in the form of a skin external preparation, specifically, ointment, emulsified cosmetic, cream, emulsion, lotion, gel, aerosol, etc. In particular, it is preferably in the form of hair rinse, hair conditioner, hair treatment, hair lotion, hair pack, hair cream, conditioning mousse, hair mousse, hair spray, shampoo, leave-on treatment, etc. .
Such preparations are prepared according to general production methods, respectively, directly or pharmaceutically acceptable carriers such as various oils, surfactants, gelling agents, preservatives, antioxidants, solvents, alcohol, water, chelate. It can be obtained by mixing and dispersing together with an agent, a thickener, an ultraviolet absorber, an emulsion stabilizer, a pH adjuster, a dye, a fragrance and the like, and then processing into a desired form. In addition, for these cosmetics, quasi drugs or pharmaceuticals, etc., plant extracts, bactericides, moisturizers, anti-inflammatory agents, antibacterial agents, cooling agents, antiseborrheic agents, etc. Can be appropriately blended within a range not impeding the effects of the present invention.

  In general, the content of the plant of the present invention or the extract thereof in the cosmetic, quasi-drug or pharmaceutical product is preferably 0.0005 to 50 wt% as a solid content concentration, and 0.001 to 20 wt%. Is more preferable.

The dosage of the above cosmetics, quasi drugs or pharmaceuticals is not particularly limited as long as the effect is obtained, and may vary according to the condition, weight, sex, age or other factors of the subject. ) As a plant per day or an extract thereof (in terms of dry matter) per person, for example, 0.01 to 1500 mg is preferable, and 0.03 to 600 mg is more preferable. The preparation can be ingested / administered according to any ingestion / administration plan, but is preferably divided into once to several times a day and continuously administered for several weeks to several months.
In addition, the application target of the above-mentioned cosmetics, pharmaceuticals or quasi drugs is not particularly limited as long as it is necessary, but a human for hair growth is preferable.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Reference Example 1 DACH1 hair tissue expression site and cell growth inhibitory action (1) Identification of DACH1 hair tissue expression site Human scalp tissue embedded with an embedding agent and frozen was microtomed to 7 μm. It was sliced into thicknesses and arranged on a MAS-coated slide glass. Then, it fixed with acetone and performed immunohistochemical staining using the following antibody.
Primary antibody:
DACH1: Anti-DACH1 antibody produced in rabbit <SIGMA-ALDRICH, HPA012672> 1/200
Ki-67: Monoclonal Mouse Anti-Human Ki-67 Antigen (Clone: MIB-1) <DAKO, IS626> Stock solution and secondary antibody
rabbit: Alexa Fluor 488 Goat Anti-rabbit IgG (highly cross-adsorbed) <Invitrogen, A-11034> 1/200
mouse: Alexa Fluor 546 Goat Anti-mouse IgG (highly cross-adsorbed) <Invitrogen, A-11030> 1/200
The results are shown in FIG.
A positive image was observed in the upper part of the hair bulb (FIG. 1). The DACH1 expression site was Ki67 (growth marker) negative, and the Ki67 positive site was DACH1 negative.

(2) Relationship between Overexpression of DACH1 and Cell Proliferation 293A cells were diluted with DMEM medium to 1 × 10 ⁴ / well, and 100 μl each was spread on a 96-well plate. After overnight incubation, transfection of the human DACH1 expression vector was performed, and the medium was changed 6 hours later. Lipofectamine 2000 (Invitrogen) was used for transfection, and the instruction manual was followed.
Note that 293A (human kidney) cells were cultured in DMEM medium (Gibco) containing 10 vol% non-immobilized FBS (Gibco), 1 vol% penicillin / streptomycin (Gibco) at 37 ° C. under 5% CO ₂ .
A human DACH1 expression vector was prepared by inserting a DACH1 PCR product (2121 bp: SEQ ID NO: 1) amplified using human hair follicle cDNA as a template into a pcDNA3.1 (+) vector (Invitrogen). Here, human hair follicle part cDNA was synthesized by extracting total RNA from human hair extracted hair root part using RNeasy Mini Kit (QIAGEN) and performing reverse transcription reaction using ThermoScript RT-PCR System (Invitrogen). . The prepared human DACH1 expression vector was sequence-analyzed and then prepared using EndoFree Plasmid Maxi Kit (QIAGEN) and used in the experiment.

24, 48 and 72 hours after transfection, 10 μl of Cell Counting Kit-8 solution (DOJINDO) was added to each well and incubated at 37 ° C. for 3 hours. Thereafter, the absorbance at 450 nm was measured with a microplate reader, and the number of viable cells was counted. The measurement time was 0.5 seconds for each well.
The obtained numerical values are shown as an average value ± standard deviation. Significant difference tests were performed on the numerical data of DACH1 (−) and DACH1 (+) at each time by the non-paired t-test method.
In addition, the case where DACH1 (-): DACH1 overexpression is not shown, and the case where DACH1 (+): DACH1 overexpression is shown is shown.
The results are shown in FIG. It was confirmed that the growth of human cultured cells (293A (human kidney) cells) was suppressed by overexpression of DACH1 (FIG. 2).

Reference Example 2 Measurement of DACH1 Function Using AP-1 Transcriptional Activity as an Index DACH1 binds to c-Jun (one of the components of AP-1 complex) and inhibits the transcriptional activity of AP-1 A DACH1 regulator search system was constructed using known information (suppressing non-patent documents 4, 5, and 6) that suppresses gene expression.
In fact, in order to confirm whether DACH1 overexpression inhibits AP-1 transcriptional activity, a human DACH1 overexpression vector and a luciferase vector into which an AP-1 binding site has been inserted are simultaneously transfected into 293A (human kidney) cells. The effect of DACH1 overexpression on AP-1 transcriptional activity was analyzed by luciferase assay.
As a result, it was possible to confirm the action of suppressing AP-1 transcriptional activity dependent on DACH1 concentration (FIG. 3).

Production Example 1 Production of Plant Extract (1) Production of Shark Extract 1.5 L of 50% aqueous ethanol solution was added to 200 g of dried roots of Shark ( Anthricus sylvestris (L.) Hoffm.) And immersed at 85 ° C. for 2 hours. This was filtered, and the filtrate was again immersed in 1.5 L of 50% ethanol aqueous solution at 85 ° C. for 1 hour. These filtrates were combined to obtain a shaku extract. When this shaku extract was concentrated, its solid content was 39 g. This solid was diluted with 50% ethanol to obtain a 1 wt% solution.
(2) Manufacture of Taiwan Hamatomoto Extract 1.5 L of 50% ethanol aqueous solution was added to 200 g of leaves of Thaiwan Hamatomoto ( Crinum asiaticum L. var. Sinicum Bak.) And immersed at 85 ° C. for 2 hours. This was filtered, and the filtrate was again immersed in 1.5 L of 50% ethanol aqueous solution at 85 ° C. for 1 hour. These filtrates were combined to obtain a Taiwan Hamamoto extract. When this Taiwan Hamaomoto extract was concentrated, its solid content was 28 g. This solid was diluted with 50% ethanol to obtain a 1 wt% solution.

Example 1 Evaluation of DACH1 Function Inhibition The DACH1 function inhibitory action of the shark extract and Taiwan Hamatomoto extract prepared in Production Example 1 was evaluated using the AP-1 transcription activity inhibitory action of DACH1 as an index.
293A cells are seeded on a 96-well plate (100 μl / well) at 1.6 × 10 ⁴ / well, and the next day, the human DACH1 expression vector, pAP1-Luc vector (Stratagene) for measuring AP-1 transcriptional activity, and internal standard PGL4.74 [hRluc / Tk] vector (Promega) was transfected.
24 hours after transfection, test substances (Shaku extract (0.01 vol%) prepared in Production Example 1 and Taiwan Hamaomoto extract (0.05 vol%)) were added, and further 24 hours later, Dual-Glo Luciferase A dual luciferase assay was performed using Assay System (Promega).
In the dual luciferase assay, after removing the medium, Dual-Glo luciferase reagent diluted 2-fold with PBS was added and stirred, and 20 minutes later, the firefly luciferase activity was measured with a microplate reader. Thereafter, an equivalent amount of Dual-Glo Stop & Glo reagent was added and stirred, and then Renilla luciferase activity was measured. Both luciferase activities were measured for 1 second. The results are shown in FIG.
As a result, the DACH1 function inhibitory action was recognized in the shark extract and the Taiwan Hamamoto extract (FIG. 4).

Example 2 Evaluation of hair elongation promoting effect The shark extract prepared in Production Example 1 was evaluated in a human hair follicle organ culture system.
Human hair follicles are isolated using a scalpel and tweezers under a stereomicroscope and in William's E medium (containing 1 vol% penicillin / streptomycin, 10 ng / mL hydrocortisone (SIGMA), 2 mM L-glutamine, 10 μg / mL insulin) Suspension culture (24 well plate) was performed at 37 ° C. and 5% CO ₂ . The test substance (Shaku extract (0.001 vol%)) was added on the day of hair follicle isolation (Day 0), and the medium was changed every 1 to 2 days.
Measurement of hair follicle elongation using photographs (every 1 to 2 days) taken with a CCD camera under a stereomicroscope was performed by measuring the length of the hair with the image analysis software and subtracting it from Day 0. did. The results are shown in FIG.
As a result, it was confirmed that the shark extract has a hair elongation promoting effect.

Claims (2)

  A hair restorer comprising as an active ingredient a plant selected from shark and Taiwan Hamaomoto or an extract thereof.   A DACH1 expression or function inhibitor comprising, as an active ingredient, a plant selected from Shark and Taiwan Hamaomoto or an extract thereof.